This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. With the advent of high mass accuracy/resolution mass analyzers suitable for the analysis of covalently modified peptides and proteins, chemical crosslinking has blossomed as a technique for mapping interacting domains and providing low resolution structures of protein complexes. The goal of this project is to extend the utility and scale of chemical crosslinking, which has generally been limited to the study of simple, binary protein interactions, to larger ensembles of cellular machinery. The largest barrier to applying structural crosslinking on this scale is the miniscule yield of intramolecular crosslinking resulting from reactions with traditional activated ester type crosslinking reagents. Reagents of this type are subject to hydrolysis, and as a result unmodified and "dead-end" modified peptides tend to predominate the reaction products of crosslinking reactions. This project encompasses: 1) the design and synthesis of new crosslinking reagents, 2) the development of methodology to enrich specifically crosslinked peptides and 3) the development of bioinformatic tools to assist in identifying crosslinked MS/MS spectra resulting from a large pool of interacting proteins.